Botulism was first described almost 200 years ago. This disease is caused by the botulinum neurotoxins (BoNT), which are seven related toxins (A-G) produced by toxigenic strains of Clostridium botulinum. These toxins are the most poisonous substances known. They act by entering neurons and cleaving proteins that mediate the exocytosis of neurotransmitters, resulting in paralysis and death. BoNTs are thought to bind to the surface of neurons via a double-receptor mechanism in which the receptor is a complex composed of gangliosides and protein(s). Identification of the toxin receptors, and the pathways that mediate entry, might provide a means to block the action of these toxins. Recent evidence indicates that members of the synaptotagmin family serve as the proteins components of the BoNT/B receptor. Synaptotagmin I and II exhibit distinct abilities to bind and mediate entry of BoNT/B. The first Aim of this proposal explores these differences with the goal of relating the structure of synaptotagmin with its ability to function as a toxin receptor. The precise mechanism by which the other BoNTs gain entry into cells is not known. Therefore, in the second Aim we will identify the pathways through which other BoNTs enter target cells. Our preliminary data indicate that different toxins enter cells via distinct pathways. In the third Aim we will further explore the means of BoNT host recognition and entry by identifying the receptor(s) that mediate internalization of BoNT/E. We have focused on this serotype because we have demonstrated feasibility using a BoNT/E affinity matrix; the long term goal is to identify receptors for additional BoNTs. Finally, in the fourth Aim we will develop FRET-based sensors that can be used to monitor BoNT activity in vitro and in living cells in real time. These sensors will make it possible to carryout high throughput screening to identify small molecules that can antagonize the action of the toxins. BoNTs are currently being used clinically to treat a variety of muscle dystonias and are also produced on large scales as potential biological weapons. The studies proposed here will provide new insights into the molecular mechanism of action of the BoNTs, and may provide a novel means to prevent poisoning by these substances.